Though combination antiretroviral therapy (ART) can potently suppress HIV replication and reduce mortality, this suppression of HIV requires lifelong administration of at least three different medicines and does not eradicate HIV, which continues to cause inflammation, ongoing damage to multiple organs systems, and reduction in life expectancy. Moreover, the worldwide prevalence of HIV continues to increase and it does not seem feasible to administer ART to all people living with HIV. Therefore, it seems imperative that we continue to pursue strategies that could lead to viral eradication or functional cure. Two main barriers to eradicatio are the presence of latent infection, as originally described in the blood, and viral reservoirs in tissues such as the gut. Despite intensive study, most of which has been performed in model systems, it is unclear what mechanisms govern latent infection in the blood. Moreover, HIV replication occurs primarily in tissues such as the gut, where most HIV-infected cells probably reside. In addition, evidence suggests important differences between gut and blood in T cell phenotypes, distribution of infected cell types, activation status, HIV transcription levels, and response to activation, suggesting that different mechanisms may regulate HIV transcription (and hence latent infection) in the gut and blood. A crucial corollary is that the gut may respond differently to therapies designed to reactivate and clear latently- infected cells in the blood. Thus, there is a critical need to understand the mechanisms that govern latent vs. productive infection in the gut and how they differ from the blood. This application will investigate the mechanisms that control HIV transcription, and hence latent infection, in blood and different gut sites in samples from HIV-infected patients using a series of novel methods. Gut tissue and cells will be obtained by endoscopic biopsies from the ileum and rectum of HIV-infected patients and uninfected controls. In aim 1, we will investigate the degree of transcriptional interference and progression through blocks to HIV transcriptional initiation and elongation in blood and gut cells from ART-treated patients using a novel panel of qRT-PCR assays specific for different HIV transcripts. In aim 2, we will use novel gut cell culture and stimulation assays to measure the degree to which different latency reversal agents can induce cellular HIV transcription and virus production from cells from gut and blood. In aim 3, we will use a novel single cell analysis assay to simultaneously quantify multiple HIV and cellular transcripts in individual gut CD4+T cells, enabling us to: 1) distinguish HIV-uninfected from infected cells as well as infected cells that do and do not transcribe HIV RNA; and 2) investigate how these populations differ in T cell phenotype and expression of human genes implicated in HIV latency and antiviral defenses. The knowledge thus gained may ultimately contribute to new and improved therapies aimed at eradication or lifelong coexistence with HIV.